The invention relates to devices for coupling liquid lines to fluidic microsystems, particularly a coupling device for liquid-tight coupling of at least one liquid line to a fluidic system, fluidic systems which are equipped with devices of this type, and methods for coupling lines to fluidic microsystems.
In biotechnology, analytics, medical research, diagnostics, and for pharmaceutical screening technologies, fluidic systems are used for handling suspended biological or synthetic samples. Miniaturized fluidic systems (micro-fluidic systems, fluidic microsystems), having typical dimensions of fluidic channels or compartments in the sub-millimeter range, are of special interest. Fluidic microsystems are particularly suitable for sample-specific single cell treatment or measurement and are equipped with microelectrode devices for this purpose if necessary. Typically, a fluidic microsystem is manufactured as a compact component (chip). The following technologies are known for charging the microsystems with the particular samples (e.g., biological cells, cell components, synthetic particles, and/or liquid media).
Firstly, receiving samples in pipette tips and applying them via tubing which is attached to the microsystem is known. Furthermore, continuously supplying microsystems with a transport or envelope stream into which the samples are introduced using pumps (e.g., syringe pumps, peristaltic pumps, piezoelectric pumps, and the like) is known. To attach tubing, providing permanent adhesive bonds, using plug-in adapters which are attached to the microsystem (see Reichle et al. “BBA”, Vol. 1459, 2000, pp. 218-229), or producing an attachment using screw bushings are known.
Permanent attachment of tubing to microsystems is disadvantageous, since for most applications flexible adaptation of the microsystem to the sample supply and separate handling of the tubing and the microsystem, e.g., for cleaning purposes, is desired. The plug-in or screw connections, in contrast, have disadvantages for producing flow, since an undesired dead volume is formed at the location of a plug-in or screw adapter, at which the flow cross-section also changes in comparison to the attached tube.
The formation of a dead volume causes multiple problems. Firstly, quantitative sample introduction or quantitative sample removal is made more difficult or prevented at low cell counts and/or small sample volumes (e.g. <10 μl, <1000 cells/μl). The applications of conventional tube couplings are restricted to microsystems in which volumes in the higher μl to ml range may be accommodated as reservoir volumes and the flow speeds and volume flows are in the range >100 μl/hour and the speeds are in the range >500 μm/second and the retrieval rate of the sample assayed is not of overwhelming interest. However, this represents a significant restriction of the field of use of conventional microsystems. Furthermore, every dead volume is connected with extended pumping times. A tube having an internal diameter of approximately 250 μm has a volume of approximately 2 μl for 1 cm of tube length. At a desired flow speed of approximately 10 μl/hour, a dwell time of approximately 10 minutes results. With an equal dead volume, an undesired extension of the pumping time accordingly results. If multiple microsystems are coupled as required by an application, unacceptable process delays result.
It is especially critical that air bubbles may form or may adhere at substrate transitions and dead volumes. Particularly in the event of discontinuous operation (“stop and go”), these lead to non-reproducible pressure changes and thus to disadvantageous movement variations of the particles or cells in the microsystem.
The dead volume is usually also connected to a change of the flow cross-section, e.g., an expansion at a connection adapter. In the event of an expansion or accordingly after a narrowing, the flow speed is reduced. Samples or sample components may settle (sedimentation). For example, undesired loss of cells or a delay may occur until the cells are flushed further. Dead volumes therefore also generate a danger due to accumulation of impurities, through which susceptibility to microbes may arise.
A coupling device for microfluidic applications is known from WO 99/63260. A hollow body is fixed on a fluidic chip, in whose end pointing toward the fluidic chip an O-ring seal is integrated via an opening in the fluidic chip. For coupling, a liquid line having a profiled external wall is plugged into the hollow body having the O-ring seal. The free end of the liquid line is pushed toward the opening until the profiled external wall of the liquid line is seated in the O-ring seal. In this state, the O-ring seal is radially compressed in the hollow body, a liquid-tight connection being formed between the liquid line and the fluidic chip.
The coupling device according to WO 99/63260 has multiple disadvantages. Firstly, the coupling device is only usable with liquid lines having a profiled line end. The line end must be processed before use if necessary (e.g., by removing material or a heat treatment). A further disadvantage arises if, by plugging the end of the liquid line into the fluidic chip, a step arises in the particular opening of the fluidic chip because of the thickness of the wall material of the liquid line, through which the dead volume having the disadvantages described above is formed. Furthermore, it is problematic that the conventional technology is designed for relatively high operating pressures (e.g., 70 bar), which are impractical, however, in fluidic microsystem technology, in which fragile glass chips are used, for example.
An essential disadvantage is that according to WO 99/63260, a good seal is achieved between the liquid line and the radially clamped O-ring. However, there is only a relatively narrow contact surface between the O-ring and the fluidic chip, whose sealing function is fulfilled unreliably because of its small dimensions. In addition, the surface of the fluidic chip is loaded unevenly. High requirements are set on the stability of the fluidic chip. If correspondingly thicker wall materials are used, disadvantages result for the applicability of optical measurement methods to the fluidic chip.
The problems cited relate not only to the coupling of tubing, but rather also generally to other connections between liquid lines (e.g., capillaries) and fluidic microsystems.
Particularly if microsystems having small intrinsic volumes are used and/or for problems in cellular biology or medicine, the following requirements may arise. Small cell counts in the range from 1 to 500 cells are to be flushed through the microsystem with a retrieval rate >70% and are to be analyzed and manipulated therein according to different criteria (e.g., size, dielectric properties, optical properties, fluorescence properties). In this case, typical pumping speeds in the range from 100 to 500 μm/seconds or pump rates in the range from 2-20 μl/hour are to be implemented. Furthermore, it is desirable for specific applications to retrieve the cells quantitatively, possibly down to individual cells. For this purpose, applications exist for isolating clones originating from individual cells and for sample preparation for single cell technologies, such as single cell PCR, single cell CE, or the like, for example.
The object of the present invention is to provide improved devices for coupling liquid lines to fluidic microsystems, using which the disadvantages of conventional coupling technologies are overcome. The devices are particularly to be distinguished by an expanded field of application, high flexibility, and improved flow-technology properties, such as minimal dead volume and avoidance of steps in the flow cross-section. The object of the present invention is also to provide improved methods for coupling liquid lines to fluidic microsystems, particularly using devices of this type.